2.1. FERRITIN
Iron is known to be an essential element of the makeup of every living organism, but may also become toxic at physiological pH values by virtue of its tending to oxidize, hydrolyze and precipitate as insoluble ferric oxide polymers. The protein ferritin, found in all living cells, is the body's means for ensuring that iron toxicity does not occur. Ferritin functions by storing iron in the cells in a soluble and readily available form. The iron stored in cells may then be mobilized whenever needed by the body, for example, for erythropoiesis.
The name "ferritin" actually encompasses a number of individual isomeric forms which are characteristic of different tissue types. Each isoferritin has 24 subunits of two distinct types, namely light subunits (L) and heavy subunits (H). These subunits differ in molecular weight, the light subunit being about 18 kDa, and the heavy subunit about 19-21 kDa. The isoferritins extracted from different tissues or organs typically exhibit different isoelectric points, with the isoelectric focusing pattern of human tissues forming a continuous spectrum; those tissues associated with high iron storage have ferritins at the basic end of the spectrum (e.g. spleen and liver), while iron poor tissues, (e.g. heart and placenta) and malignant cells have acidic ferritins. (Drysdale, Ciba Found. Symp. 51:41, 1977). The difference in isoelectric point appears to be related to the different distribution of light and heavy subunits in each type. Specifically, heavy subunit-rich ferritins are relatively acidic, and light chain rich ferritins are relatively basic (Cosell, et al., in Ferritins and Isoferritins as Biochemical Markers, p. 49-65, 1984, Elsevier). Current studies indicate that the H and L subunits are encoded by a complex group of genes, therefore suggesting that there is an even greater heterogeneity of ferritin molecules than had previously been expected.
A specific type of acidic isoferritin has been shown to be characteristic of neoplastic cells and placental cells (Drysdale and Singer, Cancer Res. 44:3352, 1974). This protein is also known as oncofetal ferritin or placental isoferritin (PLF). Human placental ferritin has been shown to be composed predominantly of a single subunit type comigrating with a liver ferritin standard on SDS-PAGE (Brown et al. Biochem. J. 182:763, 1979). However, an immunoradiometric assay performed with anti-human spleen ferritin has shown tissue specific antigenicity for PLF. (Brown et al., supra). A three subunit structure has been revealed for PLF (Moroz et al. G.I. Pat. Clin. 1:17-23, 1986). In addition to the L and H subunits characteristic of all ferritins, there is also a high molecular weight (43 kDa) subunit which appears to be unique for human placenta, and thus provides a potential site for identification of the placental isoferritin molecule as distinguished from any other type of ferritin.
2.2. PATHOLOGICAL PREGNANCY
While the majority of women who become pregnant have no substantial difficulty in carrying a child to term, there are certain conditions which commonly arise in connection with problem pregnancies. Certain visible abnormalities, such as bleeding, may be symptomatic of more serious problems, but may also be a mere irregularity which does not develop into a condition which threatens the normal pattern of gestation. On the other hand, certain conditions such as spontaneous abortion, may occur without any warning, resulting in premature termination of the pregnancy and often in the death of the fetus. Although some women, in successive pregnancies, show a pattern of difficulty with carrying a fetus to term, and can therefore be treated accordingly in advance, the development of problems is not uniformly predictable, particularly in a first pregnancy. The early identification of a woman in a high risk category would facilitate prescription of an appropriate treatment regimen, thereby increasing the chances of full-term birth of a healthy baby, as well as decreasing the possible dangers to the mother's health. Some of the more commonly occurring problems during pregnancy are toxemia, premature contractions, premature delivery, missed abortion, and spontaneous abortions or miscarriages.
A substantial number of pregnant women, perhaps as high as 7%, experience rapid weight gain, edema, and elevation of blood pressure at some time during their pregnancy. This condition, known as toxemia, results in a decrease in blood flow and in glomerular filtration, a situation which is completely the reverse of what is observed in a normal pregnancy. Because of the reduction in the glomerular filtration rate, the major problem is water retention. A particularly severe form of toxemia, eclampsia, is characterized by extreme vascular spasticity throughout the body, and clonic convulsion, followed by greatly decreased kidney output, hypertension and a general toxic condition. Toxemia of course threatens the health of the fetus, and in its more severe forms, may also threaten the mother's life.
Other problems may also arise during pregnancy which can be an indication of risk of premature delivery, or of spontaneous abortions. Many women may experience premature contractions at an early stage (as early as 16 weeks of gestation) during a pregnancy. The correlation of this symptom with a high risk of preterm delivery has not been clearly established. Similarly, abnormal bleeding can frequently occur at any time during pregnancy. This symptom may be an indication of an imminent miscarriage or a missed abortion; on the other hand, in about 50% of the cases it does not develop into anything more serious. Clearly, an early evaluation of such conditions, with some predictive significance as to risk of premature delivery or spontaneous abortion, would be of tremendous value to the clinician in both a hospital and office setting.
It has frequently been noted that something of an anomaly exists in a pregnancy being carried to full term: a fetal trophoblast which implants in the mother's uterus carries major histocompatibility antigens from both the mother and the father, and thus, except in rare circumstances, presents antigens to the maternal circulation (and immune system) which must, under normal circumstances, recognize the father's antigens as foreign. The embryo and fetus therefore stand in the position of an allograft, and, as yet, there has been no fully satisfactory explanation as to why the mother does not reject the fetus in much the same manner as a foreign skin graft would be rejected. It is clear that the embryo is protected in some way from the action of the mother's immune system. A number of mechanisms have been postulated to explain this phenomenon; among them are low levels or absence of Class II antigens on the syncytiotrophoblast which is in closest contact with the placenta, making it more likely that paternal Class I antigens will induce tolerance rather than a cytotoxic response; protection of trophoblast cells against cytotoxic lymphocytes by a barrier of negatively-charged mucopolysaccharide, or a protective effect of the physical barrier provided by the placenta.
Currently, one of the more popular theories of fetal protection is the suggestion of a general suppression of the mother's immune response. The placenta is known to secrete a variety of different products into the maternal circulation; these products include human chorionic gonadotropin (HCG) as well as substance known to have an immunoregulatory effect, such as estrogen, progesterone, corticosteroids, and pregnancy-associated growth factors (Caldwell et al. J. Immunol. 115:1249, 1975; Fabris et al. Clin. Exp. Immunol. 28:306, 1977; Baer et al. Ciba Foundation Symp. Excerpts Medica 64:293, 1979; Suteri et al. Ann. NY Acad. Sci 286:384, 1977; Monse et al. J. Immunol. 128:218, 1982). Placental isoferritin is also known to be secreted into the maternal circulation by the placenta (Brown et al. Biochem. J. 182:763, 1979). It has been suggested that some substance secreted by the placenta early in development may be responsible for inducing a general immunosuppression in the mother, thereby preventing rejection of the embryo and allowing normal full-term delivery.
In the same vein, a substantial number of irregularities which can occur during pregnancy also remain essentially unexplained as to cause. It has been suggested that some, or many of these problems may be associated with a mother's mounting an immunological response to the presence of foreign antigens on the fetus. In other words, in problem pregnancies, it is possible that the difficulties arise because of the failure of this postulated temporary immunosuppression to develop. The mother then reacts normally in response to a foreign stimulus, and various degrees of rejection of the fetus may then occur.
Evidence obtained in connection with the present invention provides the basis for suggestion of a mechanism for at least part of the physiological and immunological basis for tolerance (or non-tolerance) of the "fetal allograft" in pregnant women. It has now been discovered that a significant positive correlation exists between high serum PLF levels in pregnant women, and successful full term delivery. Similarly, abnormally low levels of PLF have been shown to be consistently associated with premature delivery, toxemia, and other pregnancy-related pathologies. Thus, low levels or absence of PLF in pregnant women can serve as marker for a potentially high risk pregnancy; detection of this state is ideally achieved by use of a PLF-specific monoclonal antibody in a novel immunoassay system, whereby early detection and diagnosis of a pathological condition can be made. This method is particularly well adapted for use in monitoring women after their first trimester of pregnancy, since levels of PLF, even in normal women, may be too low to detect prior to that time. For diagnosis of potential problems in first trimester, however, it has been discovered that detection of a relatively high level of ferritin bearing lymphocytes (FBL's) indicative of the occurrence of immunosuppression. Thus, pregnant women in the first trimester having less than about 5% of FBLs can be identified as potentially being at risk for a problem pregnancy.
The role of PLF in pregnancy does not appear to be limited to its use as a convenient marker however; there is also ample evidence to show that PLF plays an active role in the immunosuppression which appears to be a necessary event to support a normal pregnancy. Based on the observation of an apparent cause-and-effect relationship between PLF levels and immunosuppression, there is thus provided a means for treatment and prevention of actual and potentially pathological pregnancies, as well as a means for inducing immunosuppressions in those clinical situations in which production of a hyporesponsive immune state is desirable, e.g., in organ or tissue transplantation, to prevent rejection of the transplant.